AB1693 RECOMBINANT CALPROTECTIN IN A CONTROLLABLE OLIGOMERIC STATE COULD HELP TO HARMONIZE MRP-8/MRP-14 IMMUNOASSAYS
Authors: A. Ohmann, D. Guschin, J. Afonso, P. Spies, D. Tobler, T. Villiger, D. Meinel, C. B. Gerhold
Journal: Annals of the Rheumatic Diseases (2023)

<h3>Background</h3> Calprotectin is a major granulocyte and monocyte alarmin protein. It can natively occur as dimeric and tetrameric complex of Mrp-8 and Mrp-14. Although, serum calprotectin is emerging as an important biomarker for rheumatoid arthritis and juvenile idiopathic arthritis, fecal calprotectin is already established as the gold standard for diagnostics and monitoring of inflammatory bowel diseases. However, standardization of fecal calprotectin assays differs significantly among providers leading to varying clinical cut-offs. A suspected reason is that calprotectin's oligomeric states can generate different quantitative results. Yet trapping calprotectin in a distinct oligomeric state is challenging. <h3>Objectives</h3> The aim of this work was to produce pure calprotectin as calibrator material with a controllable oligomeric state. <h3>Methods</h3> The oligomeric state of a recombinant fusion protein between Mrp8 and Mrp14 that expressed solubly in E. coli was assessed by size-exclusion chromatography. Direct comparison to native calprotectin regarding antibody affinities were measured and its use as calibrator material in various immunoassay formats was tested. <h3>Results</h3> The fusion protein was purified as a calprotectin dimer mimic. Calcium addition could successfully transform calprotectin dimer into a calprotectin tetramer. Affinities to monoclonal and polyclonal calprotectin antibodies were comparable to native calprotectin. Spiking of different concentrations of recombinant calprotectin showed linear correlations in ELISA and turbidimetric assays. A perfect correlation (slope=1.009; R<sup>2</sup>=0.999) was shown when measuring 23 human serum samples based on native and recombinant calprotectin calibrators with a turbidimetric assay. <h3>Conclusion</h3> The recombinant protein can be purified in large quantities in defined oligomeric states and it shows immunological properties comparable to native calprotectin. Recombinant calprotectin presents as a promising tool to overcome the prevalent fecal calprotectin standardization problem and prevent future standardization discrepancies for serum calprotectin. <h3>REFERENCES:</h3> NIL. <h3>Acknowledgements:</h3> NIL. <h3>Disclosure of Interests</h3> Alexander Ohmann Employee of: Author is employee of BÜHLMANN Laboratories AG, Dmitrii Guschin Employee of: Author is employee of BÜHLMANN Laboratories AG, Joana Afonso Employee of: Author is employee of BÜHLMANN Laboratories AG, Peter Spies Shareholder of: Novartis, Employee of: Novartis until 1999, Daniela Tobler Employee of: I was employed in the past by Novartis. However, I am currently only employed by FHNW., Thomas Villiger Shareholder of: Yes, but only a few shares (I worked once for Novartis and Biogen…), Speakers bureau: Yes, but not for this project, Paid instructor for: Yes, but not for this project, Consultant of: Yes, but not for this project, Grant/research support from: Yes, but not for this project, Employee of: I was employed in the past by Merck, Novartis and Biogen. However, I am currently only employed by FHNW., Dominik Meinel Shareholder of: Roche shares, Employee of: regular employee at Roche 2017-2020, Christian-Benedikt Gerhold Employee of: Author is employee of BÜHLMANN Laboratories AG.